U.S. patent application number 17/258175 was filed with the patent office on 2021-05-13 for use of peptides as insecticidal agents.
The applicant listed for this patent is Institut National de Recherche Pour L'Agriculture, L'Alimentation et L'Environnement, INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE LYON. Invention is credited to PEDRO DA SILVA.
Application Number | 20210137126 17/258175 |
Document ID | / |
Family ID | 1000005401792 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210137126 |
Kind Code |
A1 |
DA SILVA; PEDRO |
May 13, 2021 |
USE OF PEPTIDES AS INSECTICIDAL AGENTS
Abstract
The present invention concerns insecticidal peptides and their
uses, in particular as plant protection agents. It also concerns
transgenic organisms, in particular transgenic plants, expressing
said peptides.
Inventors: |
DA SILVA; PEDRO; (LYON,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE LYON
Institut National de Recherche Pour L'Agriculture, L'Alimentation
et L'Environnement |
VILLEURBANNE
PARIS |
|
FR
FR |
|
|
Family ID: |
1000005401792 |
Appl. No.: |
17/258175 |
Filed: |
July 5, 2019 |
PCT Filed: |
July 5, 2019 |
PCT NO: |
PCT/FR2019/051679 |
371 Date: |
January 6, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/50 20200101;
C12N 15/8281 20130101; C12N 15/8286 20130101 |
International
Class: |
A01N 63/50 20060101
A01N063/50; C12N 15/82 20060101 C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 2018 |
FR |
1856262 |
Claims
1-17. (canceled)
18. A method of protecting a plant against an insect comprising
applying, to the plantas an insecticidal agent, a peptide selected
from the group consisting of: a peptide comprising a sequence of
amino acids selected from the group consisting of SEQ ID NO: 1, 2,
3, 4, 5 and 6, and a peptide comprising a sequence with formula
(I), having at least 80% sequence identity with one of SEQ ID NO:
1, 2, 3, 4, 5 or 6, and having an insecticidal activity, said
sequence with formula (I) being ##STR00003## in which X.sub.1
represents a sequence of 1 to 9 amino acids, X.sub.2 represents a
sequence of 2 amino acids, X.sub.3 represents a sequence of 3 amino
acids, X.sub.4 represents a sequence of 3 amino acids, X.sub.5
represents a sequence of 3 to 5 amino acids, X.sub.6 represents a
sequence of 4 and 6 amino acids, X.sub.7 represents one amino acid,
X.sub.8 represents a sequence of 9 amino acids, X.sub.9 represents
a sequence of 2 amino acids, X.sub.10 represents a sequence of 2 to
16 amino acids, or a salt of said peptide which is acceptable in
terms of plant protection.
19. The method of claim 18, wherein the peptide comprises SEQ ID
NO: 1, 2, 3, 4, 5 or 6.
20. The method of claim 18, wherein the peptide comprises SEQ ID
NO: 1, 2, 3 or 4.
21. The method of claim 20, wherein the peptide comprises SEQ ID
NO: 1.
22. The method of claim 18, in which the size of the peptide is in
the range 40 to 100 amino acids.
23. The method of claim 22, in which the size of the peptide is in
the range 40 to 70 amino acids.
24. The method of claim 18, in which the peptide is used as an
insecticidal agent against hemiptera, lepidoptera, coleoptera
and/or diptera.
25. The method of claim 24, in which the peptide is used as an
insecticidal agent against hemiptera.
26. The method of claim 18, in which the peptide is used as an
insecticidal agent against aphids.
27. The method of claim 26, wherein the aphids are Acyrthosiphon
pisum.
28. A heterologous nucleic acid comprising a nucleic acid coding
for a peptide, said nucleic acid being placed under the control of
a transcriptional promoter enabling the expression of said nucleic
acid in a cell and said peptide being: a sequence of amino acids
selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5 and
6, or a sequence of formula (I), said sequence having at least 80%
sequence identity with one of SEQ ID NO: 1, 2, 3, 4, 5 or 6 and
having an insecticidal activity, said sequence with formula (I)
being ##STR00004## in which X.sub.1 represents a sequence of 1 to 9
amino acids, X.sub.2 represents a sequence of 2 amino acids,
X.sub.3 represents a sequence of 3 amino acids, X.sub.4 represents
a sequence of 3 amino acids, X.sub.5 represents a sequence of 3 to
5 amino acids, X.sub.6 represents a sequence of 4 and 6 amino
acids, X.sub.7 represents one amino acid, X.sub.8 represents a
sequence of 9 amino acids, X.sub.9 represents a sequence of 2 amino
acids, X.sub.10 represents a sequence of 2 to 16 amino acids,
29. A method for the production of a transgenic plant, comprising
introducing a heterologous nucleic acid according to claim 28 into
a plant cell, and reconstituting said plant from said cell.
30. An entomopathogenic virus comprising a heterologous nucleic
acid of claim 28, said nucleic acid being placed under the control
of a transcriptional promoter enabling the expression of said
nucleic acid in the insect infected by the virus.
31. A non-human host cell comprising a heterologous nucleic acid of
claim 28.
32. The host cell of claim 31, in which the cell is an
entomopathogenic microorganism, an entomopathogenic bacterium or an
entomopathogenic fungus.
33. The host cell of claim 31, in which the cell is: a) a plant
cell; b) a leguminous plant or cereal plant cell; c) a leguminous
plant cell selected from the group consisting of soya, bean, pea,
chickpea, peanut, lentil, alfalfa, feva bean and carob; or d) a
cereal plant cell selected from the group consisting of wheat and
corn.
34. A transgenic plant or multicellular plant structure comprising
at least one cell as claimed in claim 31.
35. A plant protection composition comprising at least one peptide,
said at least one peptide comprising a sequence of amino acids
selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5 and
6, or a sequence with formula (I) having at least 80% sequence
identity with one of SEQ ID NO: 1, 2, 3, 4, 5 or 6, and having an
insecticidal activity, said sequence with formula (I) being
##STR00005## in which X.sub.1 represents a sequence of 1 to 9 amino
acids, X.sub.2 represents a sequence of 2 amino acids, X.sub.3
represents a sequence of 3 amino acids, X.sub.4 represents a
sequence of 3 amino acids, X.sub.5 represents a sequence of 3 to 5
amino acids, X.sub.6 represents a sequence of 4 and 6 amino acids,
X.sub.7 represents one amino acid, X.sub.8 represents a sequence of
9 amino acids, X.sub.9 represents a sequence of 2 amino acids,
X.sub.10 represents a sequence of 2 to 16 amino acids, or one of
its acceptable salts in terms of plant protection, a host cell
expressing said at least one peptide, and/or a virus comprising a
heterologous nucleic acid encoding said at least one peptide, and a
support and/or excipient which is acceptable in terms of plant
protection.
36. The plant protection composition of claim 35, further
comprising one or more additional plant protection agents.
37. The plant protection composition of claim 36, wherein the
additional plant protection agent is selected from insecticides,
bactericides, fungals, virucides, growth regulators or stimulators
of the natural defenses of the plant.
Description
[0001] The present invention relates to insecticidal peptides and
their uses, in particular as plant protection agents.
TECHNICAL PRIOR ART OF THE INVENTION
[0002] The economic impact of losses from harvests due to insect
pests is extremely important and costs billions of dollars every
year.
[0003] Currently, the protection of crops is almost exclusively
based on chemical treatments, which are being used to a greater and
greater extent, but are being widely criticized because of their
persistence in the environment and their toxicity as regards
non-target organisms such as amphibians, aquatic fauna, other
insects, or in fact the agriculturalists using such products. Thus,
the insecticides that are currently used the most against aphids,
namely neonicotinoids, will very shortly be prohibited in the
European Union because they have been shown to be hazardous to
pollinators.
[0004] In addition to these social and health concerns, it should
also be noted that the last few decades have seen the emergence of
destructive insects that are capable of resisting conventional
treatments.
[0005] The development of novel, effective insecticidal molecules
of natural origin that are not toxic to human beings and which are
more environmentally friendly appears to be indispensable in order
to limit the use of conventional insecticides obtained from
chemical synthesis.
SUMMARY OF THE INVENTION
[0006] The aim of the present invention is to provide novel
insecticidal agents which are effective, in particular against
aphids, which can be used as plant protection agents and which are
therefore suitable for replacing the current treatments using
neonicotinoids.
[0007] Thus, the present invention concerns the use, as a
pesticidal agent, preferably as an insecticidal agent, of a peptide
comprising the sequence with formula (I)
##STR00001##
in which X.sub.1 represents a sequence of 1 to 9 amino acids,
X.sub.2 represents a sequence of 2 amino acids, X.sub.3 represents
a sequence of 3 amino acids, X.sub.4 represents a sequence of 3
amino acids, X.sub.5 represents a sequence of 3 to 5 amino acids,
X.sub.6 represents a sequence of 4 and 6 amino acids, X.sub.7
represents one amino acid, X.sub.8 represents a sequence of 9 amino
acids, X.sub.9 represents a sequence of 2 amino acids, X.sub.10
represents a sequence of 2 to 16 amino acids, or a salt of said
peptide which is acceptable in terms of plant protection.
Preferably,
[0008] X.sub.1 represents the sequence (Z).sub.m-[G/S], preferably
the sequence (Z).sub.m-G, where Z is an amino acid that is selected
independently for each of the m occurrences and m is 0 or a whole
number between 1 and 8; and/or
[0009] X.sub.2 represents the sequence [F/Y]-Z, preferably the
sequence F-Z, where Z is an amino acid; and/or
[0010] X.sub.4 represents the sequence [S/D/N]-Z-Z, preferably
[S/D]-Z-Z, and more particularly preferably the sequence S-Z-Z,
where Z is an amino acid that is selected independently for each
occurrence; and/or
[0011] X.sub.5 represents the sequence (Z).sub.n-[L/V], preferably
the sequence (Z).sub.n-V, where Z is an amino acid that is selected
independently for each of the n occurrences and n is a whole number
between 2 and 4, and/or X.sub.6 represents the sequence
[G/N]-(Z).sub.p, preferably N-(Z).sub.p, where Z is an amino acid
that is selected independently for each of the p occurrences and p
is a whole number between 3 and 5; and/or
[0012] X.sub.8 represents the sequence Z-[E/Q]-Z-Z-Z-Z-Z-[D/G]-Z
(SEQ ID NO: 7), preferably the sequence Z-E-Z-Z-Z-Z-Z-D-Z (SEQ ID
NO: 8), where Z is an amino acid that is selected independently for
each occurrence; and/or
[0013] X.sub.9 represents the sequence [P/A]-[Y/H], preferably the
sequence P-Y.
In particular
[0014] X.sub.1 may represent the sequence
Z.sub.1-Z.sub.2-Z.sub.3-Z.sub.4-Z.sub.5-Z.sub.6-Z.sub.7-Z.sub.8-Z.sub.9,
where [0015] Z.sub.1 is absent or is D, and is preferably D, [0016]
Z.sub.2 is absent or is F or I, and is preferably F, [0017] Z.sub.3
is absent or is D, and is preferably D, [0018] Z.sub.4 is absent or
is P or Y, and is preferably P, [0019] Z.sub.5 is absent or is T, N
or H, and is preferably T, [0020] Z.sub.6 is absent or is E, T or
Y, and is preferably E, [0021] Z.sub.7 is absent or is F, L or I,
and is preferably F, [0022] Z.sub.8 is absent or is K, R or E, and
is preferably K, and [0023] Z.sub.9 is G or S, preferably G;
and/or
[0024] X.sub.2 may represent the sequence [F/Y]-[P/E/Q/L],
preferably F-P; and/or
[0025] X.sub.3 may represent the sequence
[I/K/R]-[E/Y/F]-[I/N/G/L], preferably I-E-I; and/or
[0026] X.sub.4 may represent the sequence
[S/D/N]-[K/T/N/R]-[Y/H/V/I], preferably S-K-Y; and/or
[0027] X.sub.5 may represent the sequence
Z.sub.10-Z.sub.11-Z.sub.12-Z.sub.13-Z.sub.14, where [0028] Z.sub.10
is absent or is E, and is preferably absent, [0029] Z.sub.11 is
absent or is Y, and is preferably absent, [0030] Z.sub.12 is A, N,
Y or H, and is preferably A, [0031] Z.sub.13 is V or K, preferably
V, [0032] Z.sub.14 is V or L, preferably V; and/or
[0033] X.sub.6 may represent the sequence
Z.sub.15-Z.sub.16-Z.sub.17-Z.sub.18-Z.sub.19-Z.sub.20 where [0034]
Z.sub.15 is N or G, preferably N, [0035] Z.sub.16 is absent or is
Y, and is preferably Y, [0036] Z.sub.17 is absent or is T, A, K or
D, preferably T, [0037] Z.sub.18 is S, I, R or E, and is preferably
S, [0038] Z.sub.19 is R, L, D, V, A or F, preferably R, and [0039]
Z.sub.20 is P, H, A or V, preferably P; and/or
[0040] X.sub.7 may represent Y, I, S or A, preferably Y.
[0041] X.sub.8 may represent the sequence
[V/I/S]-[E/Q]-[A/Y/D]-[A/H/D/R/K]-[K/S/A/R]-[E/M/Q/L]-[R/E/L/N/K]-[D/G]-[-
Q/L/H/M] (SEQ ID NO: 9), preferably V-E-A-A-K-E-R-D-Q (SEQ ID NO:
10); and/or
[0042] X.sub.9 may represent the sequence [P/A]-[Y/H], preferably
the sequence P-Y; and/or
[0043] X.sub.10 may represent the sequence
Z.sub.21-Z.sub.22-Z.sub.23-Z.sub.24-Z.sub.25-Z.sub.26-Z.sub.27-Z.sub.28-Z-
.sub.29-Z.sub.30-Z.sub.31-Z.sub.32-Z.sub.33-Z.sub.34-Z.sub.35-Z.sub.36,
where [0044] Z.sub.21 is Y, R, A, Q or P, preferably Y, [0045]
Z.sub.22 is D, T, E or N, preferably D, [0046] Z.sub.23 is absent
or is G, and is preferably absent, [0047] Z.sub.24 is absent or is
G, S, H, Q or K, and is preferably absent, [0048] Z.sub.25 is
absent or is P or A, and is preferably absent, [0049] Z.sub.26 is
absent or is A, E or S, and is preferably absent, [0050] Z.sub.27
is absent or is M, Q, E or V, and is preferably absent, [0051]
Z.sub.28 is absent or is L, M, E or V, and is preferably absent,
[0052] Z.sub.29 is absent or is M or L, and is preferably absent,
[0053] Z.sub.30 is absent or is H, and is preferably absent, [0054]
Z.sub.31 is absent or is N, Q or D, and is preferably absent,
[0055] Z.sub.32 is absent or is F, and is preferably absent, [0056]
Z.sub.33 is absent or is L, and is preferably absent, [0057]
Z.sub.34 is absent or is T or S, and is preferably absent, [0058]
Z.sub.35 is absent or is S or N, and is preferably absent, [0059]
Z.sub.36 is absent or is P, and is preferably absent.
[0060] More preferably, the peptide is selected from the group
constituted by [0061] a peptide comprising, or consisting of, a
sequence of amino acids selected from the group constituted by the
sequences SEQ ID NO: 1 to 6, and [0062] a peptide comprising, or
consisting of, a sequence with formula (I) having at least 50%,
preferably at least 80%, at least 85%, at least 90%, at least 95%,
at least 98% or at least 99% sequence identity with one of
sequences SEQ ID NO: 1 to 6, and having a pesticidal activity,
preferably an insecticidal activity.
[0063] More particularly preferably, the peptide comprises, or
consists of, a sequence of amino acids selected from the group
constituted by the sequences SEQ ID NO: 1 to 6, preferably
comprises, or consists of, a sequence of amino acids selected from
the group constituted by the sequences SEQ ID NO: 1 to 4, and more
particularly preferably comprises, or consists of, the sequence of
amino acids SEQ ID NO: 1.
[0064] The peptide preferably has a size in the range 40 to 100
amino acids, preferably in the range 40 to 70 amino acids.
[0065] The peptide may be used as an insecticidal agent against
hemiptera, lepidoptera, coleoptera and/or diptera, preferably
against hemiptera and more particularly aphids, in particular
Acyrthosiphon pisum.
[0066] The present invention also concerns a non-human host cell
comprising a heterologous nucleic acid coding for a peptide as used
in accordance with the invention, said nucleic acid being placed
under the control of a transcriptional promoter enabling the
expression of said nucleic acid in said cell.
[0067] The cell may be a plant cell, preferably a leguminous plant
or cereal plant cell, and more particularly preferably a leguminous
plant cell selected from the group constituted by soya, bean, pea,
chickpea, peanut, lentil, alfalfa, feva bean and carob or a cereal
plant cell selected from the group constituted by wheat and
corn.
[0068] The cell may also be an entomopathogenic microorganism,
preferably an entomopathogenic bacterium or fungus.
[0069] The present invention also concerns an entomopathogenic
virus comprising a heterologous nucleic acid coding for a peptide
as used in accordance with the invention, said nucleic acid being
placed under the control of a transcriptional promoter enabling the
expression of said nucleic acid in the insect infected by the
virus.
[0070] The present invention also concerns a transgenic plant or
multicellular plant structure comprising at least one cell in
accordance with the invention, as well as a method for the
production of such a transgenic plant, comprising introducing a
heterologous nucleic acid coding for a peptide as used in
accordance with the invention into a plant cell, and reconstituting
said organism from said cell.
[0071] The present invention furthermore concerns a plant
protection composition comprising at least one peptide as used in
accordance with the invention or one of its acceptable salts in
terms of plant protection, a host cell and/or a virus in accordance
with the invention, and a support and/or excipient which is
acceptable in terms of plant protection. The composition may
furthermore comprise one or more additional plant protection
agents, preferably selected from insecticides, bactericides,
fungals, virucides, growth regulators or stimulators of the natural
defenses of the plant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1: Sequence for BCR4 peptide showing the C1-05, C2-C4
and C3-C6 disulfide bridges.
[0073] FIG. 2: ESI-HRMS spectrum (A) and HPLC chromatogram (B) of
BCR4 peptide (retention time 22.5 min).
[0074] FIG. 3: Multiple alignment for members of the BCR1-2-4-5
family
DETAILED DESCRIPTION OF THE INVENTION
[0075] The inventors have identified a novel family of natural
insecticidal peptides that may be used as an alternative to
conventional chemical pesticides.
[0076] These peptides belong to a novel class of cysteine-rich
proteins known as "Bacteriocyte Cysteine Rich" (BCRs) and recently
identified in the pea aphid Acyrthosiphon pisum (Shigenobu et al.
2013). In this organism, there are seven BCRs containing 67 to 108
residues, with six or eight cysteine residues. They are coded by
orphelin genes and exclusively expressed in the bacteriocytes of
the aphids. These host cells harbor Buchnera aphidicola, the
primary symbiotic bacterium of aphids. The BCR sequences do not
exhibit any significant similarity with other sequenced proteins of
the organisms and a comparative analysis has confirmed that these
peptides are limited to the aphid line.
[0077] In the present application, the inventors have demonstrated
that the BCR4 peptide, belonging to the BCR1-2-4-5 sub-family, has
an exceptional insecticidal activity against the pea aphid with a
range of activity (5-80 .mu.M) similar to that of the AG41 peptide,
a promising entomotoxic peptide promoter of plant origin (WO
2015/087238 A1). The peptides of this family thus constitute an
effective alternative to the use of pesticides obtained by chemical
synthesis.
[0078] In accordance with a first aspect, the present invention
concerns the use, as a pesticidal agent, and more particularly as
an insecticidal agent, of a peptide comprising, or consisting of,
the sequence with formula (I)
##STR00002##
[0079] in which
[0080] X.sub.1 represents a sequence of 1 to 9 amino acids,
[0081] X.sub.2 represents a sequence of 2 amino acids,
[0082] X.sub.3 represents a sequence of 3 amino acids,
[0083] X.sub.4 represents a sequence of 3 amino acids,
[0084] X.sub.5 represents a sequence of 3 to 5 amino acids,
[0085] X.sub.6 represents a sequence of 4 to 6 amino acids,
[0086] X.sub.7 represents one amino acid,
[0087] X.sub.8 represents a sequence of 9 amino acids,
[0088] X.sub.9 represents a sequence of 2 amino acids,
[0089] X.sub.10 represents a sequence of 2 to 16 amino acids,
[0090] or a salt of said peptide which is acceptable in terms of
plant protection.
[0091] In the formula (I), the lines connecting the various
cysteine residues represent the disulfide bridges.
[0092] In the present document, the terms "peptide",
"oligopeptide", "polypeptide" and "protein" are used
interchangeably and refer to a chain of amino acids linked via
peptide linkages, irrespective of the number of amino acid residues
constituting this chain.
[0093] As used here, the term "amino acid" refers to the 20
standard natural amino acid residues (G, P, A, V, L, I, M, C, F, Y,
W, H, K, R, Q, N, E, D, S and T), to the rare natural amino acid
residues (for example hydroxyproline, hydroxylysine,
allohydroxylysine, 6-N-methyllysine, N-ethylglycine,
N-methylglycine, N-ethyl asparagine, allo-isoleucine, N-methyl
isoleucine, N-methylvaline, pyroglutamine or aminobutyric acid) and
to non-natural amino acids (for example norleucine, norvaline and
cyclohexylalanine). Preferably, this term refers to the standard 20
natural amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K,
R, Q, N, E, D, S and T).
[0094] The amino acids are represented here by their single-letter
or three-letter code in accordance with the following nomenclature:
A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E:
glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H:
histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine
(Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro);
Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T:
threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y:
tyrosine (Tyr).
[0095] The amino acids constituting the peptide used in accordance
with the invention may have the L or D configuration, preferably
the L configuration.
[0096] In accordance with one embodiment,
[0097] X.sub.1 represents the sequence (Z).sub.m-[G/S], preferably
the sequence (Z).sub.m-G, where Z is an amino acid that is selected
independently for each of the m occurrences and m is 0 or a whole
number between 1 and 8; and/or
[0098] X.sub.2 represents the sequence [F/Y]-Z, preferably the
sequence F-Z, where Z is an amino acid; and/or
[0099] X.sub.4 represents the sequence [S/D/N]-Z-Z, preferably
[S/D]-Z-Z, and more particularly preferably the sequence S-Z-Z,
where Z is an amino acid that is selected independently for each
occurrence; and/or
[0100] X.sub.5 represents the sequence (Z).sub.n-[L/V], preferably
the sequence (Z).sub.n-V, where Z is an amino acid that is selected
independently for each of the n occurrences and n is a whole number
between 2 and 4, and/or
[0101] X.sub.6 represents the sequence [G/N]-(Z).sub.p, preferably
N-(Z).sub.p, where Z is an amino acid that is selected
independently for each of the p occurrences and p is a whole number
between 3 and 5; and/or
[0102] X.sub.8 represents the sequence Z-[E/Q]-Z-Z-Z-Z-Z-[D/G]-Z
(SEQ ID NO: 7), preferably the sequence Z-E-Z-Z-Z-Z-Z-D-Z (SEQ ID
NO: 8), where Z is an amino acid that is selected independently for
each occurrence; and/or
[0103] X.sub.9 represents the sequence [P/A]-[Y/H], preferably the
sequence P-Y.
[0104] In accordance with one particular embodiment, X.sub.1
represents the sequence
Z.sub.1-Z.sub.2-Z.sub.3-Z.sub.4-Z.sub.5-Z.sub.6-Z.sub.7-Z.sub.8-Z.sub.9,
where [0105] Z.sub.1 is absent or is D, preferably is D, [0106]
Z.sub.2 is absent or is F or I, preferably is F, [0107] Z.sub.3 is
absent or is D, preferably is D, [0108] Z.sub.4 is absent or is P
or Y, preferably is P, [0109] Z.sub.5 is absent or is T, N or H,
preferably is T, [0110] Z.sub.6 is absent or is E, T or Y,
preferably is E, [0111] Z.sub.7 is absent or is F, L or I,
preferably is F, [0112] Z.sub.8 is absent or is K, R or E,
preferably is K, and [0113] Z.sub.9 is G or S, preferably G;
and/or
[0114] X.sub.2 represents the sequence [F/Y]-[P/E/Q/L], preferably
F-P; and/or
[0115] X.sub.3 represents the sequence [I/K/R]-[E/Y/F]-[I/N/G/L],
preferably I-E-I; and/or
[0116] X.sub.4 represents the sequence [S/D/N]-[K/T/N/R]-[Y/H/V/I],
preferably S-K-Y; and/or
[0117] X.sub.5 represents the sequence
Z.sub.10-Z.sub.11-Z.sub.12-Z.sub.13-Z.sub.14, where [0118] Z.sub.10
is absent or is E, preferably is absent, [0119] Z.sub.11 is absent
or is Y, preferably is absent, [0120] Z.sub.12 is A, N, Y or H,
preferably is A, [0121] Z.sub.13 is V or K, preferably V, [0122]
Z.sub.14 is V or L, preferably V; and/or
[0123] X.sub.6 represents the sequence
Z.sub.15-Z.sub.16-Z.sub.17-Z.sub.18-Z.sub.19-Z.sub.20, where [0124]
Z.sub.15 is N or G, preferably N, [0125] Z.sub.16 is absent or is
Y, preferably is Y, [0126] Z.sub.17 is absent or is T, A, K or D,
preferably T, [0127] Z.sub.18 is S, I, R or E, and is preferably S,
[0128] Z.sub.19 is R, L, D, V, A or F, preferably R, and [0129]
Z.sub.20 is P, H, A or V, preferably P; and/or
[0130] X.sub.7 represents Y, I, S or A, preferably Y; and/or
[0131] X.sub.8 represents the sequence
[V/I/S]-[E/Q]-[A/Y/D]-[A/H/D/R/K]-[K/S/A/R]-[E/M/Q/L]-[R/E/L/N/K]-[D/G]-[-
Q/L/H/M] (SEQ ID NO: 9), preferably V-E-A-A-K-E-R-D-Q (SEQ ID NO:
10); and/or
[0132] X.sub.9 represents the sequence [P/A]-[Y/H], preferably the
sequence P-Y; and/or
[0133] X.sub.10 represents the sequence
Z.sub.21-Z.sub.22-Z.sub.23-Z.sub.24-Z.sub.25-Z.sub.26-Z.sub.27-Z.sub.28-Z-
.sub.29-Z.sub.30-Z.sub.31-Z.sub.32-Z.sub.33-Z.sub.34-Z.sub.35-Z.sub.36,
where [0134] Z.sub.21 is Y, R, A, Q or P, preferably Y, [0135]
Z.sub.22 is D, T, E or N, preferably D, [0136] Z.sub.23 is absent
or is G, preferably is absent, [0137] Z.sub.24 is absent or is G,
S, H, Q or K, preferably is absent, [0138] Z.sub.25 is absent or is
P or A, preferably is absent, [0139] Z.sub.26 is absent or is A, E
or S, preferably is absent, [0140] Z.sub.27 is absent or is M, Q, E
or V, preferably is absent, [0141] Z.sub.28 is absent or is L, M, E
or V, preferably is absent, [0142] Z.sub.29 is absent or is M or L,
preferably is absent, [0143] Z.sub.30 is absent or is H, preferably
is absent, [0144] Z.sub.31 is absent or is N, Q or D, preferably is
absent, [0145] Z.sub.32 is absent or is F, preferably is absent,
[0146] Z.sub.33 is absent or is L, preferably is absent, [0147]
Z.sub.34 is absent or is T or S, preferably is absent, [0148]
Z.sub.35 is absent or is S or N, preferably is absent, [0149]
Z.sub.36 is absent or is P, preferably is absent.
[0150] In accordance with another particular embodiment,
[0151] X.sub.1 is selected from the group constituted by the
sequences G, S, YHYIES (SEQ ID NO: 11), YFES (SEQ ID NO: 12),
DFDPTEFKG (SEQ ID NO: 13), DIDPNTLRG (SEQ ID NO: 14); and/or
[0152] X.sub.2 is selected from the group constituted by the
sequences FL, FQ, FE, FP and YP; and/or
[0153] X.sub.3 is selected from the group constituted by the
sequences KYL, RYN, RFG, KYN, IEI and KEI; and/or
[0154] X.sub.4 is selected from the group constituted by the
sequences DRV, NNI, DNV, DTH, SKY; and/or
[0155] X.sub.5 is selected from the group constituted by the
sequences HKL, YKL, NKL, AVV and EYNVV (SEQ ID NO: 15); and/or
[0156] X.sub.6 is selected from the group constituted by the
sequences GDEFV (SEQ ID NO: 16), GSAA (SEQ ID NO: 17), GKRVP (SEQ
ID NO: 18), GKIDH (SEQ ID NO: 19), NYTSRP (SEQ ID NO: 20) and GASLP
(SEQ ID NO: 21); and/or
[0157] X.sub.7 represents Y, I, S or A; and/or
[0158] X.sub.8 is selected from the group constituted by the
sequences IQYKSLKGL (SEQ ID NO: 22), SQYRSLKGM (SEQ ID NO: 23),
VQYDAMNGL (SEQ ID NO: 24), IQYHSMEGL (SEQ ID NO: 25), VEAAKERDQ
(SEQ ID NO: 26) and VQDARQLDH (SEQ ID NO: 27); and/or
[0159] X.sub.9 is selected from the group constituted by the
sequences PH, PY and AY; and/or
[0160] X.sub.10 is selected from the group constituted by the
sequences PTGKASVVLHNFLTSP (SEQ ID NO: 28), ANGQAAQVLHNFLSN (SEQ ID
NO: 29), QEGHAAEELHQF (SEQ ID NO: 30), RTGSAAQMLHDFLSNP (SEQ ID NO:
31), YD and YDGGPEMLM (SEQ ID NO: 32).
[0161] In accordance with one preferred embodiment, the peptide is
selected from the group constituted by a peptide comprising, or
consisting of, a sequence of amino acids selected from the group
constituted by the sequences
TABLE-US-00001 (SEQ ID NO: 1)
DFDPTEFKGPFPTIEICSKYCAVVCNYTSRPCYCVEAAKERDQWFPYCY D, (SEQ ID NO: 2)
GPFLTKYLCDRVCHKLCGDEFVCSCIQYKSLKGLWFPHCPTGKASVVLH NFLTSP, (SEQ ID
NO: 3) YFESPFETKYNCDTHCNKLCGKIDHCSCIQYHSMEGLWFPHCRTGSAAQ MLHDFLSNP,
(SEQ ID NO: 4) DIDPNTLRGPYPTKEICSKYCEYNVVCGASLPCICVQDARQLDHWFAYC
YDGGPEMLM, (SEQ ID NO: 5)
SPFQTRYNCNNICHKLCGSAACACSQYRSLKGMWFPHCANGQAAQVLH NFLSN, and (SEQ ID
NO: 6) YHYIESPFETRFGCDNVCYKLCGKRVPCSCVQYDAMNGLWFPHCQEGH
AAEELHQFL,
[0162] and functional variants thereof.
[0163] Preferably, the peptide is selected from the group
constituted by a peptide comprising, or consisting of, a sequence
of amino acids selected from the group constituted by the sequences
SEQ ID NO: 1 to 4, and functional variants thereof. More
particularly preferably, the peptide is selected from the group
constituted by a peptide comprising, or consisting of, the sequence
of amino acids SEQ ID NO: 1, and functional variants thereof.
[0164] The term "functional variant" refers to a peptide with
formula (I) and for which the sequence differs from the parent
protein by at least one substitution, insertion or deletion but
which conserves a pesticidal activity, preferably an insecticidal
activity.
[0165] Preferably, the functional variants have at least 50%
sequence identity with the parent sequence, more particularly
preferably at least 55%, 60%, 65%, 70%, 75%, 80% or 85% sequence
identity with the parent sequence, and more particularly preferably
at least 90%, 95%, 98% or 99% sequence identity with the parent
sequence.
[0166] As used here, the term "sequence identity" or "identity"
refers to the number (%) of pairings (identical amino acid
residues) at positions originating from an alignment of two
polypeptide sequences. The sequence identity is determined by
comparing the sequences when they are aligned in a manner such as
to maximize overlapping and identity while minimizing interruptions
to the sequence. In particular, the sequence identity may be
determined using any one of the many global or local alignment
algorithms, depending on the length of the two sequences. Sequences
with similar lengths are preferably aligned using global alignment
algorithms (e.g. Needleman & Wunsch, J. Mol. Biol 48:443,
1970), which align the sequences in an optimal manner over the
entire length, while sequences with substantially different lengths
are preferably aligned using a local alignment algorithm, for
example the algorithm from Smith and Waterman (Smith and Waterman,
Adv. Appl. Math. 2:482, 1981) or the algorithm from Altschul
(Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402; Altschul
et al. (2005) FEBS J. 272:5101-5109). Alignment with the aim of
determining the percentage identity of the amino acid sequence may
be carried out using any method that is known to the person skilled
in the art, for example by using software that is available on
websites such as http://blast.ncbi.nlm.nih.gov/or
http://www.ebi.ac.uk/Tools/emboss/. The person skilled in the art
will readily be able to determine the appropriate parameters for
measuring the alignment. For the purposes of the present invention,
the values for the percentage sequence identity of the amino acids
designate values generated using the "Protein BLAST" (or Blastp)
program in which the parameters are the default parameters (Expect
threshold: 10, Word size: 6, Matrix=BLOSUM62, Gap Costs:
Existence=11, Extension=1, Conditional compositional score matrix
adjustment).
[0167] In accordance with certain embodiments, the functional
variants may differ from the parent sequence by 1 to 10, i.e. 1, 2,
3, 4, 5, 6, 7, 8, 9 to 10 substitutions, insertions and/or
deletions of amino acids, preferably by 1 to 5, i.e. 1, 2, 3, 4 or
5 substitutions, insertions and/or deletions of amino acids.
[0168] The term "substitution" as used here designates the
replacement of one amino acid residue by another selected from the
20 standard natural amino acid residues, the rare natural amino
acid residues and non-natural amino acids. Preferably, the term
"substitution" refers to the replacement of one amino acid residue
by another selected from the 20 standard natural amino acid
residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S
and T). The substitution(s) may be conservative or non-conservative
substitutions. The term "conservative substitution as used in this
document refers to a substitution of one residue of amino acid by
another which has similar chemical or physical properties (size,
charge or polarity). Examples of conservative substitutions include
(i) basic amino acids (arginine, lysine and histidine), (ii) acidic
amino acids (glutamic acid and aspartic acid), (iii) polar amino
acids (glutamine and asparagine or serine, threonine and tyrosine),
(iv) hydrophobic amino acids (methionine, leucine, isoleucine and
valine), (v) aromatic amino acids (phenylalanine, tryptophan) and
(vi) small amino acids (glycine, alanine). Preferably, the
substitutions are conservative substitutions.
[0169] In accordance with one particular embodiment, the peptide
comprises, or consists of, a sequence of amino acids selected from
the group constituted by the sequences SEQ ID NO: 1 to 6,
preferably comprises, or consists of, a sequence of amino acids
selected from the group constituted by the sequences SEQ ID NO: 1
to 4, and more particularly preferably comprises, or consists of,
the sequence of amino acids SEQ ID NO: 1.
[0170] The peptide used in accordance with the invention preferably
has a size in the range 40 to 100 amino acids, more particularly
preferably in the range 40 to 80 amino acids, and yet more
particularly preferably in the range 40 to 70 amino acids. In
accordance with one particular embodiment, the peptide used in
accordance with the invention has a size of 50 to 60 amino
acids.
[0171] The peptide used in accordance with the invention may
comprise the sequence with formula (I) fused to another protein
domain. This protein domain may, for example, be a signal sequence
or a tag.
[0172] In particular, the peptide may be a precursor that undergoes
post-translational modifications in order to obtain the nature and
active form of the peptide. It may therefore comprise a
translocation signal sequence and recognition sites and/or cleavage
sites which enables it to undergo these post-translational
modifications.
[0173] In accordance with one particular embodiment, the peptide is
a precursor of a mature pesticide or insecticidal peptide, said
precursor comprising a signal sequence for N-terminal
translocation. The signal sequence may in particular be the
sequence M-[R/K]-L-L-[Y/H]-G-F-L-I-I-M-L-T-[M/I]-[Y/H]-LS-[V/I]-Q
(SEQ ID NO: 33). The peptide may also comprise a N-terminal or
C-terminal tag that is useful for the purification or
immobilization of the peptide. Tags of this type are well known to
the person skilled in the art and include, for example, the tags
histidine (His.sub.6), FLAG, HA (epitope derived from the
hemagglutinin of the influenza virus), MYC (epitope derived from
the human proto-oncoprotein MYC) or GST
(glutathione-S-transferase). Optionally, the peptide may comprise a
site for cleavage by a protease or a chemical agent enabling this
tag to be removed.
[0174] The peptide used in accordance with the invention may have
one or more post-translational modifications and/or chemical
modifications, in particular a glycosylation, an amidation, an
acylation, an acetylation and/or a methylation.
[0175] In order to increase the resistance of the peptide to
peptidases, protective groups may be added to the C-terminal and/or
N-terminal ends. As an example, the protective group at the
N-terminal end may be an acylation or an acetylation and the
protective group at the C-terminal end may be an amidation or an
esterification. The action of the proteases may also be
counteracted, for example, by using amino acids with a D
configuration or pseudo-peptide linkages replacing the
"conventional" CONH peptide linkages, such as CHOH--CH.sub.2, NHCO,
CH.sub.2--O, CH.sub.2CH.sub.2, CO--CH.sub.2, N--N, CH.dbd.CH,
CH.sub.2NH, and CH.sub.2--S.
[0176] The invention also encompasses the use of salts of said
peptide, preferably salts which are acceptable in terms of plant
protection. A salt that is acceptable in terms of plant protection
is a salt that has no notable toxicity in the dose in which it is
used, having regard to the plant, the environment or human beings.
As an example, the salts could be salts with acceptable mineral
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid
and phosphoric acid; salts with acceptable organic acids such as
acetic acid, citric acid, maleic acid, malic acid, succinic acid,
ascorbic acid or tartaric acid; salts with acceptable mineral bases
such as sodium, potassium, calcium, magnesium or ammonium salts; or
salts with organic bases that have a salifiable nitrogen. In
accordance with certain preferred embodiments, the salt is an
ammonium sulfate. These salts are routinely used and their
preparation methods are well known to the person skilled in the
art.
[0177] The invention concerns the use of the peptide as described
above as a pesticidal agent, and more particularly as an
insecticidal agent, preferably for plant protection
applications.
[0178] As used here, the term "pesticide" refers to a compound
intended to combat organisms that are considered to be harmful.
When these organisms are insects, the term "insecticide" is
used.
[0179] As used here, the term "plant protection application" refers
to an action intended to protect plants and cultivated products
against harmful organisms, in particular insects, and/or to ensure
the conservation of cultivated products. The term "cultivated
products" means products which are gathered or harvested, in
particular vegetables, fruit, flowers or grain, and the transformed
products that are derived from them, in particular flour.
[0180] Preferably, the peptide is used as an insecticidal agent in
a plant protection treatment.
[0181] The insecticidal activity of the peptide can easily be
tested by the person skilled in the art, in particular by observing
the survival of insect larvae, for example pea aphid larvae, on a
liquid medium containing the peptide to be tested. This routine
technique is illustrated in the experimental section of the present
application. Preferably, a peptide is considered to have an
insecticidal activity when it has a LT.sub.50 (lethal time at which
mortality of individuals is 50%) of less than 5 days on insect
larvae, preferably pea aphid larvae, at a concentration of 100
.mu.M, preferably a concentration of 50 .mu.M.
[0182] The insecticidal activity of the peptide with formula (I) is
preferably against insect pests selected from the group constituted
by hemiptera, lepidoptera, coleoptera and diptera.
[0183] Examples of hemiptera include, without being limited
thereto, species of aphids such as Acyrthosiphon pisum, Aphis
gossypii, Aulacorthum solani, Macrosiphum euphorbiae, Myzus
persicae and Rhopalosiphum padi, cochineal insect species such as
Planococcus citri, whitefly species such as Trialeurodes
vaporariorum and Bemisia tabaci, or bug species such as Lygocoris
pabulinus, Liocoris tripustulatus, Lygus rugulipennis and
Corythucha ciliata.
[0184] Examples of lepidoptera include, without being limited
thereto, caterpillars of several butterfly species such as
Chrysodeixis chalcites, Mamestra brassicae, Spodoptera exigua,
Clepsis spectrana, Cacoecimorpha prombana, Duponchelia fovealis,
Tuta absoluta, Cydia pomonella, Lobesia botrana and Eupoecillia
ambiguella.
[0185] Examples of coleoptera include, without being limited
thereto, weevil species such as Sitophilus spp. And wireworm
species such as Agriotes spp.
[0186] Examples of diptera include, without being limited thereto,
species of flies such as Sciaridae spp., Musca domestica, Stomoxys
calcitrans and Liriomyza spp.
[0187] In accordance with certain preferred embodiments, the
peptide is used as an insecticidal agent against one or more
hemiptera, and more particularly one or more aphids, in particular
Acyrthosiphon pisum.
[0188] The peptide described above may be used alone or in
combination with one or more other active agents. The additional
active agent or agents may in particular be selected from the other
peptides described above and with formula (I) and other pesticidal,
insecticidal or plant protection agents, preferably with one or
more other insecticidal agents.
[0189] Preferably, the peptide is used in combination with one or
more other insecticidal peptides. In accordance with one particular
embodiment, the peptide is used in combination with the
insecticidal peptide AG41 (WO 2015/087238, Table II).
[0190] The present invention also concerns an expression cassette
comprising a nucleic acid coding for a peptide as described above
and with formula (I), operationally linked to a heterologous
transcriptional promoter.
[0191] The term "expression cassette" designates a nucleic acid
construct comprising a coding region and a regulatory region which
are operationally linked. The expression "operationally linked"
indicates that the elements are combined in a manner such that the
expression of the coding sequence is under the control of the
transcriptional promoter. Typically, the sequence for the promoter
is placed upstream of the gene of interest, at a distance therefrom
which is compatible with control of the expression.
[0192] The cassette may furthermore comprise other regulatory
elements such as a transcription terminator, the sequence for a
transit peptide and/or an activating sequence termed an
"enhancer".
[0193] As used here, the term "heterologous transcriptional
promoter" refers to a promoter which, because of its nature, is not
associated with a nucleic acid coding for the peptide. The cassette
in accordance with the invention is thus a recombinant
cassette.
[0194] The transcriptional promoter can readily be selected by the
person skilled in the art as a function of the nature of the host
cell in which expression is envisaged. In particular, this promoter
may be a prokaryotic or eukaryotic, systemic or tissue-specific,
inducible or constitutive promoter.
[0195] Preferably, the transcriptional promoter is a promoter
allowing the expression of the peptide in a host plant cell or an
entomopathogenic microorganism.
[0196] Alternatively, the cassette is intended to be inserted into
a viral vector and the transcriptional promoter is a promoter
enabling the expression of the peptide in a host cell infected by
said vector, preferably a cell of an insect pest.
[0197] The expression cassettes in accordance with the invention
may be constructed using conventional molecular biological
techniques which are well known to the person skilled in the
art.
[0198] The present invention also concerns an expression vector
comprising an expression cassette in accordance with the invention.
This expression vector may be used to transform a host cell and
enable expression of the nucleic acid coding for a peptide as
described above and with formula (I) in said cell.
[0199] The vector may be a DNA or an RNA, which may or may not be
circular, or it may be single-stranded or double-stranded. It is
advantageously selected from a plasmid, a phage, a phagemid, a
virus, a cosmid and an artificial chromosome.
[0200] The person skilled in the art will readily be able to select
the most appropriate vector, in particular having regard to the
target host cell, the desired level of expression and the desired
stability, the mode selected for introducing the vector into the
host cell, etc.
[0201] The vector may also comprise elements enabling it to be
selected in the host cell such as, for example, a gene for
resistance to an antibiotic or to a herbicide, or a selection gene
ensuring complementation of the respective gene that has been
deleted in the genome of the host cell. Elements of this type are
well known to the person skilled in the art and have been widely
described in the literature.
[0202] The vectors in accordance with the invention may furthermore
comprise an origin of replication and/or a sequence allowing
targeted insertion into the genome of the host cell.
[0203] The vectors in accordance with the invention may be
constructed using conventional molecular biological techniques
which are well known to the person skilled in the art.
[0204] In accordance with a particular embodiment, the vector is a
viral vector, preferably an entomopathogenic virus. Thus, the
present invention concerns an entomopathogenic virus comprising a
nucleic acid coding for a peptide as described above and with
formula (I), said nucleic acid being placed under the control of a
transcriptional promoter enabling expression of said nucleic acid
in the host cell infected by the virus, the host cell preferably
being an insect cell,
[0205] The entomopathogenic virus may be selected from the
Baculoviridae, the Reoviridae, the Poxviridae, the Iridoviridae,
the Parvoviridae, the Picornoviridae, and the Rhabdoviridae.
[0206] Preferably, the entomopathogenic virus belongs to the
Baculoviridae family.
[0207] The transcriptional promoter can readily be selected by the
person skilled in the art as a function of the target cell.
[0208] The present invention also concerns the use of an expression
cassette or an expression vector in accordance with the invention
in order to transform or transfect a cell. The host cell may be
transformed/transfected in a transitory or stable manner and the
cassette or the vector may be contained in the cell in the form of
an episome or in the chromosomal form.
[0209] The present invention concerns a non-human host cell
comprising a cassette or an expression vector in accordance with
the invention.
[0210] It also concerns a host cell comprising a heterologous
nucleic acid (which is not naturally present in the cell) coding
for a peptide as described above and with formula (I). Said nucleic
acid is preferably placed under the control of a transcriptional
promoter enabling the expression of said nucleic acid in the cell.
This promoter may be an endogenous promoter (naturally present in
the cell) or a heterologous promoter (which is not naturally
present in the cell).
[0211] The host cell may in particular be selected in order to act
as a propagation vector for the peptide with formula (I) or for the
purposes of production of the peptide.
[0212] The host cell may be a prokaryotic or eukaryotic cell. The
host cell may in particular be a plant cell, an animal cell, a
fungal cell or a bacterium.
[0213] In accordance with one embodiment, the host cell is an
animal cell. In particular, the animal cell could be selected from
the cells of mammals such as COS or CHO cells, the cells of insects
such as Sf9, Sf21 or Hi5 cells, or the cells of protozoa or
nematodes.
[0214] The cells of protozoa and of nematodes are preferably
selected from entomopathogenic families, in particular the families
used in biological control such as, for example; the families of
the Amoebidae and Nosematidae protozoa or the nematode species
Steinermatidae and Heterorhabditidae.
[0215] In accordance with another embodiment, the host cell is a
cell of a microorganism, preferably a cell of an entomopathogenic
microorganism and more particularly preferably an entomopathogenic
bacterial cell or fungal cell.
[0216] Many bacteria have been described as being entomopathogenic.
In particular, the bacteria may be selected from bacteria from the
Bacillaceae, Enterobacteriaceae and Pseudomonaceae families.
Preferably, the bacterium is selected from Bacillus thurengiensis
and Bacillus sphaericus.
[0217] Similarly, many fungi have been described as being
entomopathogenic. In particular, the fungus may be selected from
fungi from the genuses Beauveria, for example Beauvaria bassiana,
Metharizium, for example Metharizium anisopliae, Verticillium,
Erynia, Hirsutella, Entomophtora and Entomophaga.
[0218] In accordance with yet another embodiment, the host cell is
a plant cell, preferably a leguminous plant or cereal plant
cell.
[0219] The plant cell may be a leguminous plant cell selected from
the group constituted by soya, bean, pea, chickpea, peanut, lentil,
alfalfa, feva bean and carob.
[0220] The plant cell may also be a cereal plant cell selected from
the group constituted by wheat and corn.
[0221] The plant cell may also be a Nicotiana, for example
Nicotiana sylvestris or Nicotiana tabacum.
[0222] The techniques for introducing a heterologous nucleic acid
into these various types of cells (plant cell, animal cell, fungal
cell or bacterial cell) are well known to the person skilled in the
art.
[0223] In accordance with another aspect, the present invention
also concerns a multicellular transgenic organism comprising a host
cell in accordance with the invention, i.e. a cell comprising a
heterologous nucleic acid coding for a peptide as described above
and with formula (I), in particular comprising a cassette or an
expression vector in accordance with the invention.
[0224] In accordance with one embodiment, the organism is a
protozoon or a nematode. In accordance with one preferred
embodiment, the organism is a transgenic plant or a multicellular
plant structure As used here, the term "multicellular plant
structure" refers to portions of a plant such as the flowers,
seeds, leaves, stems, fruit, pollen, tubers, wood or multicellular
structures such as the calluses, organs of the plant or immature
embryos (for example explants used for transgenesis).
[0225] The reconstitution of a transgenic plant or of a
multicellular plant structure as defined above from a transfected
or transformed plant cell falls is within the scope of routine
techniques which are well known to the person skilled in the
art.
[0226] The peptide may be expressed ubiquitously (in all of the
tissues of the plant) or only in certain tissues or organs, in
particular the tissues or organs which are the targets of the pest
organism.
[0227] All of the embodiments concerning the other aspects of the
invention are also envisaged in this aspect.
[0228] The present invention also concerns a method for the
production of a transgenic plant or of a multicellular plant
structure comprising a host cell in accordance with the invention,
said method comprising the introduction, into a plant cell, of a
nucleic acid coding for a peptide as described above and with
formula (I), of an expression cassette or of a vector in accordance
with the invention, and the reconstitution of said plant or
structure from said cell.
[0229] Optionally, the method may also comprise a step for
selecting plants or structures containing the nucleic acid, the
cassette or the vector. This selection may be carried out using any
method known to the person skilled in the art, in particular by DNA
amplification methods.
[0230] All of the embodiments concerning the other aspects of the
invention are also envisaged in this aspect.
[0231] The present invention also concerns a method for the
production of a peptide as described above and with formula (I),
comprising the transformation or transfection of a cell by a
nucleic acid coding for said peptide or a cassette or expression
vector in accordance with the invention; culturing the
transfected/transformed cell; and harvesting the peptide produced
by said cell. Methods for the production of recombinant peptides
are well known to the person skilled in the art.
[0232] The cell used for the production of the peptide may be a
prokaryotic or a eukaryotic cell.
[0233] In accordance with one particular embodiment, the cell is a
bacterium, in particular E. coli. Preferably, the bacterium used is
capable of carrying out oxidative folding of the peptide, such as,
for example, a strain of SHuffle E. coli (Lobstein et al. Microbial
Cell Factories. 2012; 11:56).
[0234] The present invention also concerns a method for the
production of a peptide as described above and with formula (I),
comprising obtaining a transgenic plant in accordance with the
invention and expressing the peptide, culturing this plant and
harvesting the peptide produced by said plant.
[0235] Preferably, the transgenic plant is a Nicotiana, for example
Nicotiana sylvestris or Nicotiana tabacum.
[0236] Methods for the production of recombinant peptides from
transgenic plants are well known to the person skilled in the
art.
[0237] The present invention also concerns a method for the
production of a peptide as described above and with formula (I),
comprising insertion of a nucleic acid coding for said peptide, of
an expression cassette or an expression vector in accordance with
the invention in an in vitro expression system, also termed
acellular, and harvesting the peptide produced by said system. Many
systems for in vitro or acellular expression are available
commercially and the use of these systems is well known to the
person skilled in the art.
[0238] The present invention also concerns a plant protection
composition comprising at least one peptide as described above and
with formula (I), a host cell or a transgenic organism in
accordance with the invention and/or a virus in accordance with the
invention, and an excipient which is acceptable in terms of plant
protection. The invention furthermore concerns the use of a peptide
as described above and with formula (I), a host cell or a
transgenic organism in accordance with the invention and/or a virus
in accordance with the invention, for the preparation of a plant
protection composition.
[0239] The plant protection composition may be of any type. It may
be an aqueous or hydro-alcoholic solution, a paste, a gel, in
particular an aqueous gel (or hydrogel), a glue, a foam, a
water-in-oil emulsion or an oil-in-water emulsion, a multiple
emulsion, a micro-emulsion or nano-emulsion, a micellar solution, a
suspension, or a colloid. The plant protection composition may also
be solid, for example in the form of a powder or granules which can
be applied directly by "dusting" or which have to be dissolved or
dispersed in a suitable solvent before application.
[0240] The term "excipient which is acceptable in terms of plant
protection" means an excipient that does not have any notable
toxicity, in the dose in which it is used, as regards the plant,
the environment and human beings. Excipients that can be used in
the plant protection compositions are well known to the person
skilled in the art and include, inter alia, diluents and fillers,
wetting agents, surfactants, for example ionic, amphoteric or
non-ionic surfactants, dispersing agents, thickening agents,
gelling agents, agents allowing the controlled release of the
active agents, for example encapsulation agents or micellar agents
such as phospholipids, thixotropic adjuvants, colorants,
antioxidants, preservatives, stabilizers, film-forming agents,
vehicles, in particular solvents such as water and lower alcohols,
oils of animal, vegetable or mineral origin, resins, waxes, rosin,
latex, gums such as gum Arabic, anti-foaming agents, and adhesive
agents.
[0241] The plant protection composition in accordance with the
invention may furthermore comprise one or more additional plant
protection agents. The additional plant protection agent or agents
may, for example, be selected from insecticides, bactericides,
fungals, virucides, growth regulators or stimulators of the natural
defenses of the plant such as elicitors.
[0242] The galenical form and the excipients of the plant
protection composition in accordance with the present invention can
readily be selected by the person skilled in the art and
essentially depend on the infestation to be treated and/or the mode
of administration.
[0243] The present invention also concerns the use of a peptide as
described above and with formula (I) or of a plant protection
composition in accordance with the invention, for the treatment or
prevention of infestation of a plant and/or a cultivated product by
pest organisms, preferably by insects, the method comprising
bringing the peptide as described above and with formula (I) into
contact with said plant, cultivated product or pest organism.
[0244] All of the references cited in this description are
incorporated into the present application by reference. Other
characteristics and advantages of the invention will become
apparent from the examples below, which are given by way of
non-limiting illustration.
Examples
Method and Apparatus
Synthesis and Purification of BCR4 Peptide
[0245] Ten milligrams of non-functional reduced unrefined BCR4
peptide (DFDPTEFKGPFPTIEICSKYCAVVCNYTSRPCYCVEAAKERDQWFPYCYD; SEQ ID
NO: 1) was obtained from Proteogenix (Strasbourg, France).
Oxidative folding of the peptide in vitro was carried out using the
protocol described above (cf. Da Silva and al, 2009). In brief, the
oxidative folding was carried out by incubating the reduced peptide
(10 mg) in a deoxygenated buffer containing 0.1 mM of oxidized
glutathione (10 equiv.), 1 mM de glutathione (100 equiv.), 1 mM of
EDTA, 100 mM TRIS, pH 8.5, at 20.degree. C., for 48 h in an
atmosphere of argon (final concentration of peptide 30 .mu.M). The
reaction was acidified by adding TFA (200 .mu.L) and the unrefined
mixture was purified by semi-preparative HPLC in order to produce
pure BCR4 (2.2 mg, i.e. 22% yield). The purity of the BCR4 peptide
was evaluated by RP-HPLC, its mass was obtained by high resolution
mass spectrometry (ESI-HRMS). The concentration of peptide was
determined by measuring the surface area of its RP-HPLC peak at 214
nm.
Insecticide Tests
[0246] The experiments were carried out on aphids from the
parthenogenetic clone of the pea aphid LL01 which contained only
the primary symbiont B. aphidicola. The aphids were bred and kept
on feva bean plants (Vicia faba L., Aquadulce variety) under
climate-controlled conditions (temperature 21.+-.1.degree. C.,
relative humidity 70%, photoperiod of 16 h), in order to obtain
aphids which reproduced solely by viviparous parthenogenesis. In
order to reduce the variability linked to the use of individuals at
different stages of development, the aphids were obtained from a
synchronized brood. For this, adult aphids that were ready to lay
were placed on feva bean plants. After 24 hours, the young larvae
were recovered and placed on an artificial medium which contained
or did not contain the peptide of interest. The manipulation was
carried out with a negative control composed solely of artificial
medium and a positive control representing one of the
concentrations of the range being studied with the aphids. The
range being studied comprised different media containing the BCR4
peptide studied at various concentrations which varied from 5 to 80
.mu.M. The experiments were carried out on thirty larvae per
condition divided into three distinct groups of ten
individuals.
Statistical Analyses
[0247] All of the data regarding the mortality of the aphids were
analyzed statistically by standard survival processing. All of the
BCR4 concentrations were analyzed separately in a parametric
survival analysis with a normal logarithmic adjustment.
Analyses of Sequences
[0248] The orthologous sequences of the BCR proteins were recovered
using a combination of TBLASTN and BLASTP (Johnson and al, 2008)
against the genomes of aphids available in the AphidBase database
(Legeai et al., 2010) and the entire non-redundant proteins,
nucleotides and EST database from NCBI. The BCR proteins underwent
multiple sequence alignments using the MUSCLE program (Edgar et
al., 2004).
Results
Synthesis and Purification of BCR4 Peptide
[0249] 2.2 mg of pure BCR4 peptide was obtained by a chemical
synthesis in the solid phase followed by oxidative folding in order
to form the C1-C5, C2-C4 and C3-C6 disulfide bridges (FIG. 1).
[0250] The purity of the BCR4 peptide was evaluated using RP-HPLC
and its mass was obtained by high resolution mass spectrometry
(ESI-HRMS): the theoretical mass calculated for BCR4 was 5891.5400
gmol.sup.-1; the experimental mass obtained was 5891.5437
gmol.sup.-1 (FIG. 2A). The concentration of peptide was determined
by measuring the surface area of its RP-HPLC peak at 214 nm (FIG.
2B).
Insecticide Tests
[0251] The measurement of the insecticidal activity of BCR4 was
carried out by incorporating increasing doses of peptides into the
nutrient medium for the aphids and monitoring of their mortality
was carried out over 7 days. A very significant dose-response over
the survival time for the aphids was observed (TLSO: lethal time at
which mortality of individuals is 50%) (Table 1).
TABLE-US-00002 TABLE 1 Toxicity of BCR4 peptide on the pea aphid
Acyrthosiphon pisum. TL50 in days, with confidence intervals,
obtained by survival analysis with a normal logarithmic adjustment.
BCR4 Concentration (.mu.M) peptide 80 40 35 27.5 20 10 5 TL50 1.16
1.76 1.94 3.24 3.48 11.3 >20 (days) [0.98- [1.27- [1.47- [2.22-
[2.28- [4.16- 1.37] 2.42] 2.57] 4.72] 5.31] 30.8]
[0252] The dose-response effect commenced very early, from the
first day following ingestion, with a high mortality. This range of
activity (5-80 .mu.M) was similar to that of the peptide AG41, an
entomotoxic promoter of plant origin (cf. WO 2015/087238).
Sequence Analysis
[0253] By means of an exhaustive search of the nucleotide and
protein databases from the NCBI coupled with a specific inspection
of sequences for aphid species available in the AphidBase (Legeai
et al., 2010), the inventors recovered a total of 32 BCR sequences.
All of these sequences were identified in aphid species. The 32 BCR
species were used to align the amino acid sequences and, according
to the consensus motifs obtained following these alignments, it
appears that the BCR sequences can be grouped into four
sub-families comprising the sequences (i) BCR1-2-4-5, (ii) BCR3,
(iii) BCR6 and (iv) BCR8 (Table 2).
TABLE-US-00003 TABLE 2 Characteristics of four BCR sub-families.
Number of Orthologs identified in cysteine aphids for which genome
residues Consensus motif.sup.a has been sequenced.sup.b BCR1-2-4-5
6 C(3)C(3-5)C(4-6)C(1)C(13)C Apis (4), Dnox (1) BCR3 6
C(3)C(6)C(21-25)C(6)C(4)C Agly (1), Apis (1), Dnox (2), Mcer (1),
Mper (1), Rpad (1) BCR6 8 C(15)C(3)C(3)C(10)C(10)C(1)CC Apis (1),
Dnox (2), Mcer (1), Mper (1) BCR8 6 C(3-4)C(10)C(17)C(1)CC Agly
(1), Apis (1), Dnox (1), Mcer (1), Mper (1), Rpad (1) .sup.aThe
number of residues between the cysteines is noted between
parentheses. .sup.bAbbreviations: Agly, Aphis glycines; Apis,
Acyrthosiphon pisum; Dnox, Diuraphis noxia; Mcer, Myzus cerasi;
Mper, Myzus persicae; Rpad, Rhopalosiphum padi.
[0254] The peptides BCR1 (SEQ ID NO: 2), BCR2 (SEQ ID NO: 3), BCR4
(SEQ ID NO: 1) and BCRS (SEQ ID NO: 4) of Acyrthosiphon pisum, as
well as two peptides from Acyrthosiphon kondoi (SEQ ID NO: 6) and
from Diuraphis noxia (SEQ ID NO: 5), belong to the sub-family
BCR1-2-4-5 which had a strictly conserved consensus motif (FIG.
3).
BIBLIOGRAPHIC REFERENCES
[0255] Da Silva P et al., 2009. Biopolymers 92(5): 436-444. [0256]
Edgar R C et al., 2004. MUSCLE: multiple sequence alignment with
high accuracy and high throughput. Nucleic Acids Res. 32,
1792-1797. [0257] Johnson M et al., 2008. NCBI BLAST: a better web
interface. NCBI BLAST: a better web interface. Nucleic Acids Res.
2008 Jul. 1; 36 (Web Server issue):W5-9. Legeai F et al., 2010.
AphidBase: a centralized bioinformatic resource for annotation of
the pea aphid genome. Insect Mol. Biol. 19, 5-12. [0258] Shigenobu
and Stern. Proc. R. Soc. B. 2013 280, 20121952.
Sequence CWU 1
1
33150PRTAcyrthosiphon pisum 1Asp Phe Asp Pro Thr Glu Phe Lys Gly
Pro Phe Pro Thr Ile Glu Ile1 5 10 15Cys Ser Lys Tyr Cys Ala Val Val
Cys Asn Tyr Thr Ser Arg Pro Cys 20 25 30Tyr Cys Val Glu Ala Ala Lys
Glu Arg Asp Gln Trp Phe Pro Tyr Cys 35 40 45Tyr Asp
50255PRTAcyrthosiphon pisum 2Gly Pro Phe Leu Thr Lys Tyr Leu Cys
Asp Arg Val Cys His Lys Leu1 5 10 15Cys Gly Asp Glu Phe Val Cys Ser
Cys Ile Gln Tyr Lys Ser Leu Lys 20 25 30Gly Leu Trp Phe Pro His Cys
Pro Thr Gly Lys Ala Ser Val Val Leu 35 40 45His Asn Phe Leu Thr Ser
Pro 50 55358PRTAcyrthosiphon pisum 3Tyr Phe Glu Ser Pro Phe Glu Thr
Lys Tyr Asn Cys Asp Thr His Cys1 5 10 15Asn Lys Leu Cys Gly Lys Ile
Asp His Cys Ser Cys Ile Gln Tyr His 20 25 30Ser Met Glu Gly Leu Trp
Phe Pro His Cys Arg Thr Gly Ser Ala Ala 35 40 45Gln Met Leu His Asp
Phe Leu Ser Asn Pro 50 55458PRTAcyrthosiphon pisum 4Asp Ile Asp Pro
Asn Thr Leu Arg Gly Pro Tyr Pro Thr Lys Glu Ile1 5 10 15Cys Ser Lys
Tyr Cys Glu Tyr Asn Val Val Cys Gly Ala Ser Leu Pro 20 25 30Cys Ile
Cys Val Gln Asp Ala Arg Gln Leu Asp His Trp Phe Ala Tyr 35 40 45Cys
Tyr Asp Gly Gly Pro Glu Met Leu Met 50 55553PRTDiuraphis noxia 5Ser
Pro Phe Gln Thr Arg Tyr Asn Cys Asn Asn Ile Cys His Lys Leu1 5 10
15Cys Gly Ser Ala Ala Cys Ala Cys Ser Gln Tyr Arg Ser Leu Lys Gly
20 25 30Met Trp Phe Pro His Cys Ala Asn Gly Gln Ala Ala Gln Val Leu
His 35 40 45Asn Phe Leu Ser Asn 50657PRTAcyrthosiphon kondoi 6Tyr
His Tyr Ile Glu Ser Pro Phe Glu Thr Arg Phe Gly Cys Asp Asn1 5 10
15Val Cys Tyr Lys Leu Cys Gly Lys Arg Val Pro Cys Ser Cys Val Gln
20 25 30Tyr Asp Ala Met Asn Gly Leu Trp Phe Pro His Cys Gln Glu Gly
His 35 40 45Ala Ala Glu Glu Leu His Gln Phe Leu 50
5579PRTArtificial Sequenceamino acid sequence represented by
"X8"misc_feature(1)..(1)Xaa can be any naturally occurring amino
acidMISC_FEATURE(2)..(2)X is E or Qmisc_feature(3)..(7)Xaa can be
any naturally occurring amino acidMISC_FEATURE(8)..(8)X is D or
Gmisc_feature(9)..(9)Xaa can be any naturally occurring amino acid
7Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 589PRTArtificial
Sequenceamino acid sequence represented by
"X8"misc_feature(1)..(1)Xaa can be any naturally occurring amino
acidmisc_feature(3)..(7)Xaa can be any naturally occurring amino
acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino
acid 8Xaa Glu Xaa Xaa Xaa Xaa Xaa Asp Xaa1 599PRTArtificial
Sequenceamino acid sequence represented by
"X8"MISC_FEATURE(1)..(1)x is V, I or SMISC_FEATURE(2)..(2)x is E or
QMISC_FEATURE(3)..(3)x is A, Y or DMISC_FEATURE(4)..(4)x is A, H,
D, R or KMISC_FEATURE(5)..(5)x is K, S, A or RMISC_FEATURE(6)..(6)x
is E, M, Q or LMISC_FEATURE(7)..(7)x is R, E, L, N or
KMISC_FEATURE(8)..(8)x is D or GMISC_FEATURE(9)..(9)x is Q, L, H or
M 9Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5109PRTArtificial
Sequenceamino acid sequence represented by "X8" 10Val Glu Ala Ala
Lys Glu Arg Asp Gln1 5116PRTArtificial Sequenceamino acid sequence
represented by "X1" 11Tyr His Tyr Ile Glu Ser1 5124PRTArtificial
Sequenceamino acid sequence represented by "X1" 12Tyr Phe Glu
Ser1139PRTArtificial Sequenceamino acid sequence represented by
"X1" 13Asp Phe Asp Pro Thr Glu Phe Lys Gly1 5149PRTArtificial
Sequenceamino acid sequence represented by "X1" 14Asp Ile Asp Pro
Asn Thr Leu Arg Gly1 5155PRTArtificial Sequenceamino acid sequence
represented by "X5" 15Glu Tyr Asn Val Val1 5165PRTArtificial
Sequenceamino acid sequence represented by "X6" 16Gly Asp Glu Phe
Val1 5174PRTArtificial Sequenceamino acid sequence represented by
"X6" 17Gly Ser Ala Ala1185PRTArtificial Sequenceamino acid sequence
represented by "X6" 18Gly Lys Arg Val Pro1 5195PRTArtificial
Sequenceamino acid sequence represented by "X6" 19Gly Lys Ile Asp
His1 5206PRTArtificial Sequenceamino acid sequence represented by
"X6" 20Asn Tyr Thr Ser Arg Pro1 5215PRTArtificial Sequenceamino
acid sequence represented by "X6" 21Gly Ala Ser Leu Pro1
5229PRTArtificial Sequenceamino acid sequence represented by "X8"
22Ile Gln Tyr Lys Ser Leu Lys Gly Leu1 5239PRTArtificial
Sequenceamino acid sequence represented by "X8" 23Ser Gln Tyr Arg
Ser Leu Lys Gly Met1 5249PRTArtificial Sequenceamino acid sequence
represented by "X8" 24Val Gln Tyr Asp Ala Met Asn Gly Leu1
5259PRTArtificial Sequenceamino acid sequence represented by "X8"
25Ile Gln Tyr His Ser Met Glu Gly Leu1 5269PRTArtificial
Sequenceamino acid sequence represented by "X8" 26Val Glu Ala Ala
Lys Glu Arg Asp Gln1 5279PRTArtificial Sequenceamino acid sequence
represented by "X8" 27Val Gln Asp Ala Arg Gln Leu Asp His1
52816PRTArtificial Sequenceamino acid sequence represented by "X10"
28Pro Thr Gly Lys Ala Ser Val Val Leu His Asn Phe Leu Thr Ser Pro1
5 10 152915PRTArtificial Sequenceamino acid sequence represented by
"X10" 29Ala Asn Gly Gln Ala Ala Gln Val Leu His Asn Phe Leu Ser
Asn1 5 10 153012PRTArtificial Sequenceamino acid sequence
represented by "X10" 30Gln Glu Gly His Ala Ala Glu Glu Leu His Gln
Phe1 5 103116PRTArtificial Sequenceamino acid sequence represented
by "X10" 31Arg Thr Gly Ser Ala Ala Gln Met Leu His Asp Phe Leu Ser
Asn Pro1 5 10 15329PRTArtificial Sequenceamino acid sequence
represented by "X10" 32Tyr Asp Gly Gly Pro Glu Met Leu Met1
53319PRTArtificial SequenceSignal sequenceMISC_FEATURE(2)..(2)x is
R or KMISC_FEATURE(5)..(5)x is Y or HMISC_FEATURE(14)..(14)x is M
or IMISC_FEATURE(15)..(15)x is Y or HMISC_FEATURE(18)..(18)x is V
or I 33Met Xaa Leu Leu Xaa Gly Phe Leu Ile Ile Met Leu Thr Xaa Xaa
Leu1 5 10 15Ser Xaa Gln
* * * * *
References